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Abstract:

One molecule of the amino acid selected from the five kinds of amino
acids consisting of cysteine, lysine, histidine, phenylalanine, and
glycine is interposed between a self-assembled monolayer and a molecule
of a protein. A method for immobilizing an protein on a self-assembled
monolayer includes the following steps (a) and (b) in this order: a step
(a) of preparing a substrate including one molecule of an amino acid and
the self-assembled monolayer and a step (b) of supplying the protein to
the substrate to form a peptide bond represented by a predetermined
chemical formula as a result of reaction between the carboxyl group of
the one molecular of the amino acid and the amino group of the protein.

Claims:

1. A method for immobilizing a protein on a self-assembled monolayer, the
method comprising: a step (a) of preparing a substrate comprising one
molecule of an amino acid and the self-assembled monolayer, wherein the
one molecule of the amino acid is bound to the self-assembled monolayer
through a peptide bond represented by the following chemical formula (I):
##STR00011## where R represents a side chain of the one molecule of the
amino acid, the one molecule of the amino acid is selected from the group
of amino acids consisting of cysteine, lysine, histidine, phenylalanine,
and glycine; and a step (b) of supplying the protein to the substrate to
form a peptide bond represented by the following chemical formula (II) as
a result of reaction between a carboxyl group of the one molecule of the
amino acid and an amino group of the protein: ##STR00012## where R
represents the side chain of the one molecule of the amino acid.

2. The method according to claim 1, wherein the step (a) comprises: a
step (a1) of preparing the substrate comprising the self-assembled
monolayer on a surface thereof, the self-assembled monolayer having a
carboxyl group at one end; and a step (a2) of supplying the one molecule
of the amino acid to form the peptide bond represented by the chemical
formula (I) as a result of reaction between the carboxyl group of the one
end of the self-assembled monolayer and an amino group of the one
molecule of the amino acid.

3. The method according to claim 1, further comprising, between the step
(a) and the step (b): a step (ab) of activating a carboxyl group of the
one molecule of the amino acid with a mixture of N-Hydroxysuccinimide and
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.

4. The method according to claim 2, further comprising, between the step
(a1) and the step (a2): a step (a1a) of activating the carboxyl group of
the self-assembled monolayer with a mixture of N-Hydroxysuccinimide and
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.

5. The method according to claim 1, wherein the chemical formula (II) is
represented by the following chemical formula (III): ##STR00013## where
R represents the side chain of the one molecule of the amino acid.

6. A sensor comprising: a self-assembled monolayer; one molecule of an
amino acid; and a protein, wherein: the one molecule of the amino acid is
interposed between the self-assembled monolayer and the protein, the
protein is bound to the self-assembled monolayer through two peptide
bonds represented by the following chemical formula (II): ##STR00014##
where R represents a side chain of the one molecule of the amino acid,
and the one molecule of the amino acid is selected from the group of
amino acids consisting of cysteine, lysine, histidine, phenylalanine, and
glycine.

7. The sensor according to claim 6, wherein the chemical formula (II) is
represented by the following chemical formula (III): ##STR00015## where
R represents the side chain of the one molecule of the amino acid.

8. A method for detecting or quantifying a target substance contained in
a sample with use of a sensor, the method comprising: a step (a) of
preparing the sensor comprising a self-assembled monolayer, one molecule
of an amino acid, and a protein, wherein the one molecule of the amino
acid is interposed between the self-assembled monolayer and the protein,
the protein is bound to the self-assembled monolayer through two peptide
bonds represented by the following chemical formula (II): ##STR00016##
where R represents a side chain of the one molecule of the amino acid,
and the one molecule of the amino acid is selected from the group of
amino acids consisting of cysteine, lysine, histidine, phenylalanine, and
glycine; a step (b) of supplying the sample to the sensor to bind the
target substance to the protein; and a step (c) of detecting the target
substance bound in the step (b), or quantifying the target substance
contained in the sample from an amount of the target substance bound in
the step (b).

9. The method according to claim 8, wherein the chemical formula (II) is
represented by the following chemical formula (III): ##STR00017## where
R represents the side chain of the one molecule of the amino acid.

10. The method according to claim 1, wherein the protein does not include
Protein A, streptavidin, glucose oxidase, antibody or albumin.

11. The sensor according to claim 6, wherein the protein does not include
Protein A, streptavidin, glucose oxidase, antibody or albumin.

12. The method according to claim 8, wherein the protein does not include
Protein A, streptavidin, glucose oxidase, antibody or albumin.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation of International application No.
PCT/JP2011/007239, with international filling date of Dec. 22, 2011,
which claims priority of Japanese Patent Application No. 2011-151573,
filed on Jul. 8, 2011, the contents of all of which are hereby
incorporated by reference.

[0002] Further, it is noted that International Patent Publication Nos.
WO2011/089903, WO2012/029202, WO2012/053138, WO2012/168988 and
WO2013/005269 are commonly owned by the Assignee of the present
application.

BACKGROUND

[0003] The present disclosure relates to a method for immobilizing a
protein on a self-assembled monolayer.

[0004] A biosensor is used to detect or quantify a target substance
contained in a sample. Some biosensors include protein capable of binding
to the target substance to detect or quantify the target substance. More
particularly, a biosensor for detecting or quantifying an antigen
includes an antibody capable for binding specifically to the antigen.
Similarly, biosensors for detecting or quantifying biotin and glucose
include streptavidin and glucose oxidase, respectively.

[0005] When a sample containing the target substance is supplied to the
biosensor including protein capable of binding to the target substance,
the target substance is bound to the protein to detect or quantify the
target substance.

[0119] of the corresponding Japanese Publication). FIG. 2
shows a biosensor disclosed in FIG. 7 of Patent Literature 1.

[0007] According to the description regarding FIG. 7 of WO00/04382, the
biosensor is used for screening an activity of a biomolecule. The
biosensor includes a monolayer 7, an affinity tag 8, an adaptor molecule
9, and a protein 10. The monolayer 7 is composed of a self-assembled
monolayer represented by chemical formula: X--R--Y (see, Page 24, lines
23-26, Page 25, lines 3-20, Page 25, line 27-Page 26, line 13, and Page
26, lines 14-22 of WO00/04382; or paragraphs

[0080],

[0082],

[0084] and

[0085] of the corresponding Japanese Publication). Examples of X, R, and
Y are HS--, an alkane, and a carboxyl group, respectively (see, Page 25,
lines 3-20, Page 25, lines 27-Page 26, line 13, and Page 28, lines 21-23
of WO00/04382; or paragraphs

[0084],

[0085], and

[0095] of the
corresponding Japanese Publication).

BRIEF SUMMARY

Technical Problem

[0008] In order to improve the detection sensitivity or the quantification
accuracy of the target substance, it is required to increase an amount of
protein to be immobilized on the biosensor.

[0009] The present inventor has discovered that the amount of the
immobilized protein per unit area was increased significantly by binding
one molecule amino acid selected from the group consisting of cysteine,
lysine, histidine, phenylalanine, and glycine to a self-assembled
monolayer and then immobilizing protein. The present subject matter has
been provided on the basis of the discovery.

[0010] The purpose of the present disclosure is to provide a method for
increasing an amount of protein to be immobilized on the self-assembled
monolayer, and a sensor with the protein immobilized in accordance with
the same method.

Solution to Problem

[0011] A method for immobilizing a protein on a self-assembled monolayer
includes the following steps. Step (a) is a step of preparing a substrate
including one molecule of an amino acid and the self-assembled monolayer.
The one molecule of the amino acid is bound to the self-assembled
monolayer through a peptide bond represented by the following chemical
formula (I):

##STR00001##

where R represents the side chain of one molecule of the amino acid. The
one molecular of the amino acid is selected from the five kinds of amino
acids consisting of cysteine, lysine, histidine, phenylalanine, and
glycine. Step (b) is a step of supplying the protein to the substrate to
form a peptide bond represented by the following chemical formula (II) as
a result of reaction between the carboxyl group of the one molecule of
the amino acid and the amino group of the protein:

##STR00002##

where R represents the side chain of the one molecule of the amino acid.

[0012] In one embodiment, the step (a) may include the following steps
(a1) and (a2). Step (a1) is a step of preparing the substrate comprising
the self-assembled monolayer on the surface thereof, the self-assembled
monolayer having a carboxyl acid at one end. Step (a2) is a step of
supplying the one molecule of the amino acid to form the peptide bond
represented by the chemical formula (I) as a result of reaction between
the carboxyl group of the one end of the self-assembled monolayer and the
amino group of the one molecule of the amino acid.

[0013] In one embodiment, the method may further include, between the step
(a) and the step (b), a step (ab) of activating the carboxyl group of the
one molecule of the amino acid with a mixture of N-Hydroxysuccinimide and
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.

[0014] In one embodiment, the method may further includes, between the
step (a1) and the step (a2), a step (a1a) of activating the carboxyl
group of the self-assembled monolayer with a mixture of
N-Hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride.

[0015] In one embodiment, the chemical formula (II) may be represented by
the following chemical formula (III):

##STR00003##

where R represents the side chain of the one molecule of the amino acid.

[0016] One aspect of the present disclosure is a sensor including a
self-assembled monolayer, one molecule of an amino acid, and a protein.
The one molecule of the amino acid is interposed between the
self-assembled monolayer and the protein, and the protein is bound to the
self-assembled monolayer through two peptide bonds represented by the
following chemical formula (II):

##STR00004##

where R represents the side chain of the one molecule of the amino acid.
The one molecule of the amino acid is selected from the five kinds of
amino acids consisting of cysteine, lysine, histidine, phenylalanine, and
glycine.

[0017] In one embodiment, the chemical formula (II) may be represented by
the following chemical formula (III):

##STR00005##

where R represents the side chain of the one molecule of the amino acid.

[0018] One aspect of the present disclosure is a method for detecting or
quantifying a target substance contained in a sample with use of a
sensor. The method includes the following steps. Step (a) is a step of
preparing the sensor including a self-assembled monolayer, one molecule
of an amino acid, and a protein. The one molecule of the amino acid is
interposed between the self-assembled monolayer and the protein, and the
protein is bound to the self-assembled monolayer through two peptide
bonds represented by the following chemical formula (II):

##STR00006##

where R represents the side chain of the one molecule of the amino acid.
The one molecule of the amino acid is selected from the five kinds of
amino acids consisting of cysteine, lysine, histidine, phenylalanine, and
glycine. Step (b) is a step of supplying the sample to the sensor to bind
the target substance to the protein. Step (c) is a step of detecting the
target substance bound in the step (b), or quantifying the target
substance contained in the sample from the amount of the target substance
bound in the step (b).

[0019] In one embodiment, the chemical formula (II) may be represented by
the following chemical formula (III):

##STR00007##

where R represents the side chain of the one molecule of the amino acid.

Advantageous Effect

[0020] The present subject matter can increase significantly the amount of
the protein to be immobilized per unit area.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows an exemplary schematic view of a method according to
one embodiment of the present disclosure.

[0022] FIG. 2 corresponds to FIG. 7 of WO00/04382.

[0023] FIG. 3 shows a schematic view of a method according to the prior
art.

DETAILED DESCRIPTION

[0024] The embodiment of the present disclosure is described below with
reference to FIG. 1.

[0025] FIG. 1 shows an exemplary method according to the present
disclosure for immobilizing protein on a self-assembled monolayer.

[0026] Preferably, a substrate 1 is a gold substrate. An example of the
gold substrate is a substrate having gold uniformly on its surface.
Specifically, the gold substrate may be a substrate having a gold film
formed by a sputtering method on the surface of glass, plastic, or
SiO2.

[0027] First, the substrate 1 is immersed into a solvent containing an
alkanethiol. Preferably, the substrate is washed before immersed. The
alkanethiol has a carboxyl group at the end thereof. It is preferable
that the alkanethiol has the carbon number within the range from six to
eighteen. Thus, a self-assembled monolayer 2 is formed on the substrate
1.

[0028] The preferred concentration of the alkanethiol is approximately 1
mM to 10 mM. The solvent is not limited to, as long as it dissolves the
alkanethiol. An example of the preferred solvent is ethanol, dimethyl
sulfoxide (hereinafter, referred to as "DMSO"), and dioxane. The
preferred immersing period is approximately 12 to 48 hours.

[0029] Next, an amino acid 3 is supplied to the self-assembled monolayer
2. The carboxyl group (--COOH), which is located at the top end of the
self-assembled monolayer 2, reacts with an amino group (--NH2) of
the amino acid 3 to form a peptide bond represented by the following the
chemical formula (I):

##STR00008##

where R represents the side chain of the one molecule of the amino acid.

[0030] In the chemical formula (I), one molecule of the amino acid 3 binds
to the self-assembled monolayer 2.

[0031] The amino acid 3 is selected from five kinds of amino acids
consisting of cysteine, lysine, histidine, phenylalanine, and glycine. In
other words, in the chemical formula (I), R is the side chain of these
five kinds of amino acids.

[0032] When the amino acid 3 is supplied to the self-assembled monolayer
2, two or more kinds of amino acids may be supplied simultaneously. In
other words, when a solution containing the amino acid 3 is supplied to
the self-assembled monolayer 2, the solution may contain two or more
kinds of the amino acids 3. In light of uniform bind of the protein to
the amino acid 3, which is described later, it is preferred that the
solution contains a sole kind of amino acid.

[0033] Subsequently, protein 4 is supplied. The 5'-terminal amino group of
the protein 4 reacts with the carboxyl group of the amino acid 3. The
amino group of the lysine included in the protein also reacts with the
carboxyl group of the amino acid 3. Thus, two peptide bonds represented
by the following chemical formula (II) are formed to obtain a sensor:

##STR00009##

where R represents the side chain of the one molecule of the amino acid.

[0034] One molecule of the protein 4 has only one N-terminus (the start of
the protein terminated by an amino acid with a free amine group),
corresponding to the 5' end of mRNA encoding the protein, whereas the one
molecule of the protein 4 has a lot of lysine groups having a free amine
group. Therefore, almost all of the chemical formula (II) is represented
more specifically by the following chemical formula (III):

##STR00010##

where R represents the side chain of the one molecule of the amino acid.

[0035] The obtained sensor is used for detecting or quantifying the target
substance contained in the sample.

EXAMPLES

[0036] The following examples and comparative examples describe the
present subject matter in more detail.

Comparative Example A 1

[0037] As shown in FIG. 3, Protein A was bound directly with an amide
coupling reaction to a carboxyl group located at the top end of
self-assembled alkanethiol formed on the gold surface to immobilize the
Protein A. The procedure and the results were described below. It is
well-known that Protein A is a protein which constitutes five percent of
the cell wall of staphylococcus aureus and is abbreviated as "SpA".

[Preparation of a Sample Solution]

[0038] A sample solution of 16-Mercaptohexadecanoic acid with final
concentration of 10 mM was prepared. The solvent thereof was ethanol.

[Formation of a Self-Assembled Monolayer]

[0039] A gold substrate (available from GE healthcare company, BR-1004-05)
with gold vapor-deposited on glass was used as a substrate 1. The
substrate 1 was washed for ten minutes with a piranha solution containing
concentrated sulfuric acid and 30% hydrogen peroxide water. The volume
ratio of the concentrated sulfuric acid to the 30% hydrogen peroxide
water contained in the piranha solution was 3:1.

[0040] Subsequently, the gold substrate was immersed in the sample
solution for 18 hours to form a self-assembled monolayer on the surface
of the gold substrate. Finally, the substrate 1 was washed with pure
water and dried.

[Immobilization of Protein]

[0041] As protein, Protein A was bound to the carboxyl acid group located
at the top end of the 16-Mercaptohexadecanoic acid which formed the
self-assembled monolayer to immobilize the Protein A.

[0042] Specifically, the carboxyl acid group located at the top end of the
16-Mercaptohexadecanoic acid was activated with use of 35 microliters of
a mixture of 0.1M NHS (N-Hydroxysuccinimide) and 0.4M EDC
(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride).
Subsequently, 35 microliters of the Protein A (40 ug/ml) was added at the
flow rate of 5 microliters/minute. Thus, the carboxyl acid of the
16-Mercaptohexadecanoic acid was coupled with the amino group of the
Protein A.

Example A1

[0043] Experiment was conducted similarly to the comparative example A1
except that glycine was supplied as the one molecule of the amino acid
between the formation of the self-assembled monolayer and the
immobilization of the Protein A. The procedure and the results are
described below.

[Immobilization of Amino Acid (Glycine)]

[0044] Glycine was bound with the carboxyl group located at the top end of
the 16-Mercaptohexadecanoic acid which formed the self-assembled
monolayer 2 to immobilize the glycine.

[0045] Specifically, after the carboxyl group was activated similarly to
the comparative example A1, 35 microliters of 0.1M glycine (pH: 8.9) was
added at the flow rate of 5 microliters/minute. Thus, the carboxyl group
of 16-Mercaptohexadecanoic acid was coupled with the amino group of the
glycine.

[Immobilization of Protein]

[0046] Subsequently, Protein A was bound to the carboxyl group of the
glycine to immobilize the Protein A. Specifically, after the carboxyl
group of the glycine was activated similarly to the above, 35 microliters
of Protein A (concentration: 250 micrograms/ml) was added at the flow
rate of 5 microliters/minute. Thus, the carboxyl group was coupled with
the 5'-terminal amino acid of the Protein A or the amino group of the
lysine included in the Protein A.

[Comparison of the Immobilization Amounts]

[0047] The immobilization amounts in the example A1 and in the comparative
example A1 were measured with use of an SPR device, Biacore 3000
(available from GE healthcare company).

[0048] The term "immobilization amount" means the amount of the protein
immobilized per unit area.

[0060] A skilled person would understand the followings from Table 1 to
Table 5.

[0061] When the one molecule of the amino acid selected from the five
kinds of amino acids consisting of cysteine, lysine, histidine,
phenylalanine and glycine is interposed between the self-assembled
monolayer and the protein, the immobilization amount of the protein per
unit area is increased, compared to the case where the one molecule of
the amino acid selected from other fifteen kinds of the amino acid is
used or to the case where one molecule of the amino acid is not used.

INDUSTRIAL APPLICABILITY

[0062] The present subject matter can increase significantly the amount of
the protein to be immobilized per unit area. This improves the
sensitivity or the accuracy of the biosensor. The biosensor may be used
for an inspection or a diagnosis which requires the detection or the
quantification of the target substance contained in the living sample
derived from a patient at a clinical practice.

[0063] In the present patent application, Protein A, streptavidin, glucose
oxidase, antibody and albumin may be excluded from the term "protein"
used in the claims.